Various workers have proposed an inverse relationship between the amount of tetrahydropterin (BH4) bound to nitric oxide synthase and the extent to which activity of the enzyme is stimulated by added BH4. Critical assessment of the validity of this proposal requires nitric oxide synthase that is free of BH4. Treatment of the enzyme with acid ammonium sulfate or charcoal (procedures commonly used to resolve enzyme-cofactor complexes) di not remove BH4 from the enzyme. Nitroblue tetrazolium, an oxidant of BH4 i solution, did not oxidize enzyme-bound BH4. These observations suggest tha BH4 is bound in a hydrophobic pocket that is sequestered from the aqueous environment. They further indicate contrary to the proposal of other workers, that enzyme-bound BH4 is not an intermediary electron carrier in the "diaphorase" activity of nitric oxide synthase, i.e., in the transfer o electrons from NADPH to a dye such as nitroblue tetrazolium. Surprisingly, we found that addition to the assay of the reduced form of the dye, dichlorophenol indophenol, results in a marked increase in the stimulation (up to 40-fold) by added BH4. The chemical nature of enzyme-bound biopteri under these conditions is now being examined. The most promising approach to clarifying the role of BH4 in nitric oxide synthase has been provided by studies with substrate amounts of enzyme. Under these conditions, in the absence of NADPH, arginine is converted to a compound tentatively characterized as hydroxyarginine; no citrulline is formed. Synthesis of hydroxyarginine is dependent on Ca2+ and calmodulin, and stimulated by BH4. Thus, NADPH appears not to be the immediate electron donor in hydroxyarginine synthesis, but is required for further metabolism of this intermediate to the end products nitric oxide and citrulline. Experiments are underway to determine the immediate electron donor required for conversion of arginine to hydroxyarginine.